Thermo Scientific ABsolute Fast QPCR Low ROX Mix

Transcription

1 Thermo Scientific ABsolute Fast QPCR Low ROX Mix Description ABsolute Fast QPCR Low ROX Mix has been developed to quantify DNA and cdna*. With the exception of primers and template, this 2X mix contains all the components required to perform a rapid, sensitive and reproducible QPCR reaction: - Thermo-Fast TM DNA Polymerase, a chemically modified hot-start version of Thermoprime Taq DNA Polymerase, which prevents non-specific amplification during the reaction set-up. This enzyme requires an activation step at 95 C for 5 minutes. - Proprietary reaction buffer which provides highly sensitive, specific and consistent fluorescence readings for real-time and end-point analysis. This buffer has been optimized for MgCl2 and enhancers to improve amplification across a wide range of templates including plant DNA and GC rich fragments. It contains an inert blue dye to assist in the visualization of the ABsolute Fast QPCR Low ROX Mix after aliquoting into the reaction well. - dntps, including dttp to improve reaction sensitivity and efficiency compared to dutp. - ROX, passive reference dye for normalization of data. Kit Contents Vial Pack Size (cap color) A B ABsolute Fast QPCR Low ROX Mix (2X) 2 x 1.25ml (clear) 16 x 1.25ml (clear) Cycler & Probe Compatibility ABsolute Fast QPCR Low ROX Mix is compatible for use with any probe system and QPCR cyclers requiring low ROX dye levels, including ABI PRISM 7500 (including Fast-Block) and Stratagene Mx4000, Mx3000P, Mx3005P. For RNA templates, we recommend our Verso cdna Synthesis Kit (AB-1453/B) for the reverse transcription step. 1

2 INFORMATION Thermo-Fast TM DNA Polymerase The enzyme requires an activation step at 95 C for 5 minutes. Thermo-Fast TM has 5' to 3' polymerization and exonuclease activity but lacks 3' to 5' exonuclease activity (proofreading). Blue Dye This proprietary inert blue dye allows quick and easy visualization of the amount of the mix in the well, minimizing aliquoting errors. It does not interfere with the QPCR reaction and is only available in master mix format. ROX Dye ROX is an internal passive reference dye used to normalize the fluorescent reporter signal generated in QPCR. The concentration of ROX in the final 1X reaction is 25 nm. Storage Conditions Store at -20 C until ready for use. ABsolute Fast QPCR Low ROX Mix is stable for a minimum of 12 months. The reagents can be stored at 4 C for up to 3 months. Avoid repeated freeze thawing. The ROX dye is light sensitive; exposure should be minimized. Shipped on ice within the UK and on dry ice for international and within the US. Additional Info - The use of disposable gloves, DNase and RNase free filter tips and plastics is recommended. - The recommended amplicon length is in the range of 60 to 300 bp although optimal results may be obtained with amplicons <150bp. - As best performance is achieved with dttp, the ABsolute Fast QPCR Low ROX Mix contains a nucleotide mix with dttp instead of dutp. 2

3 DIRECTIONS FOR USE Tips and Protocol Thaw the reagents on ice. Mix and spin down the solutions before use to recover the maximum amount. Do not vortex the ABsolute Fast QPCR Low ROX Mix. Briefly centrifuge to avoid bubbles within the wells, as these will interfere with the fluorescence. Always include a no template control (NTC). Example of Reaction Mix preparation for a 25 µl final reaction: Reaction Mix Volume Final Concentration ABsolute Fast QPCR Low ROX Mix (2X) 12.5 µl 1X Forward primer (10 µm) a 1 µl 400 nm Reverse primer (10 µm) a 1 µl 400 nm Probe Variable nm Water (PCR grade) b Variable Template (DNA or cdna) c 1-5 µl <250 ng/reaction Total volume 25 µl Example of a fast QPCR thermal cycling program: Temp. Time Number of cycle Enzyme activation 95 C 5 min 1 cycle Denaturation 95 C 1 sec d Annealing/Extension 60 C 20 sec d 40 cycles Notes a Start by using 400 nm of each primer. If optimization is required, try stepping the primer concentration up or down in 25nM increment between 100 nm and 500 nm final concentration. b The volume of the total reaction should be completed up to 25 µl with water. c The volume of template to add to the QPCR reaction can be adjusted as required. Routinely, only 1 µl of template DNA or cdna should be added to reduce carryover of PCR inhibitors. This volume can be increased up to 5 µl for low copy number templates. d The fast thermal protocol shown may require optimization for best results. Some assays may require longer dwell times for efficient amplification. If so, increasing denaturation and annealing/extension dwell times in 5 second increment may be beneficial. Conversely, the dwell times may be decreased for some amplicons offering even faster QPCR results. Please note that it may be necessary to redesign the assay with a shorter amplicon (<150bp) to support fast thermal cycling. 3

4 Quality control ABsolute Fast QPCR Low ROX Mix is tested functionally using QPCR. The product must demonstrate linearity of amplification over a specified serial dilution of human genomic DNA under fast cycling conditions. Ordering Information AB-4329/A ABsolute Fast QPCR Low ROX Mix 200 x 25 µl rxns AB-4329/B ABsolute Fast QPCR Low ROX Mix 1,600 x 25 µl rxns AB-4330/A ABsolute Fast QPCR Low ROX Mix 400 x 25 µl rxns AB-4330/B ABsolute Fast QPCR Low ROX Mix 4,000 x 25 µl rxns Related Products Cat. No. Description Quantity AB-0600/W Thermo-Fast TM 96 Non-Skirted, white * 25 plates AB-1100/W Thermo-Fast TM 96 PCR Detection Plate, white * 25 plates AB-1400/W Thermo-Fast TM 96 PCR Detection Plate Mark II, white * 25 plates AB-1170 ABsolute TM QPCR Seal (adhesive seal) 50 sheets AB-0812 Clear Seal Diamond (heat seal) 100 sheets AB-0866 Ultra Clear Cap Strips (8 caps) 120 strips * For Cycler compatibility and other color choices, see our latest catalogue or visit For technical information or troubleshooting contact Thermo Scientific Genomics Tech Support: Troubleshooting: Phone North America (US, Canada, Central/South America) Europe (EU, Middle East, Africa) Other Countries dharmacondistributors +1 (800) (+) A license to perform the patented 5 Nuclease Process for research is obtained by the purchase of (i) both Authorized 5' Nuclease Core Kit and Licensed Probe, (ii) a Licensed 5 Nuclease Kit, or (iii) license rights from Applied Biosystems. This product is an Authorized 5 Nuclease Core Kit. Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 6,127,155, 5,677,152, 5,773,258, and claims outside the US corresponding to US Patent No. 4,889,818, 5,079,352, 5,210,015 and 5,487,972. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research. Separate purchase of a Licensed Probe would convey rights under the applicable claims of US Patents Nos. 5,538,848, 5,723,591, 5,876,930, 6,030,787, 6,258,569 and 5,804,375 (claims 1-12 only), and corresponding claims outside the United States. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by 4

5 implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Use of this product in a passive reference method is covered by the following U.S. Patent: 5,928,907 (claim numbers 12-24, 27-28) and corresponding patent claims outside the US. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product in a passive reference method for the purchaser s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Revised January, All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. Literature Code: AB-4329-v3 5